1. Field of the Invention
This invention relates to fluorescence in situ hybridization.
2. Background Art
In situ hybridization (ISH) is a powerful and versatile tool for the detection and localization of nucleic acids (DNA and RNA) within cell or tissue preparations. By the use of labeled DNA or anti-sense RNA probes, the technique provides a high degree of spatial information in locating specific DNA or RNA sequences within individual cells or chromosomes. ISH is widely used for research and potentially for diagnosis in the areas of prenatal genetic disorders, and molecular cytogenetics. In the general area of molecular biology, ISH is used to detect gene expression and over-expression, to map genes, to identify sites of gene expression, to localize target genes, and to identify and to localize various viral and microbial infections. Currently, the application of the ISH technology research is being expanded into tumor diagnosis, preimplantation genetic diagnosis for in vitro fertilization, evaluation of bone marrow transplantation, and analysis of chromosome aneuploidy in interphase and metaphase nuclei.
In ISH, labeled nucleic acids (DNA or anti-sense RNA) are hybridized to chromosomes or mRNAs in cells which are immobilized on microscope glass slides (In Situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In Situ Hybridization: In Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); and In Situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England (1992)). Numerous non-isotopic systems have been developed to visualize labeled DNA probes including; a) fluorescence-based direct detection methods, b) the use of digoxigenin-and biotin-labeled DNA probes coupled with fluorescence detection methods, and c) the use of digoxigenin-and biotin-labeled DNA probes coupled with antibody-enzyme detection methods. When fluorescence-labeled nucleic acid (DNA or RNA) probes are hybridized to cellular DNA or RNA, the hybridized probes can be viewed directly using a fluorescence microscope. By using multiple nucleic acid probes with different fluorescence colors, simultaneous multicolored analysis (i.e., for multiple genes or RNAs) can be performed in a single step on a single target cell. Fluorochrome-directly-labeled nucleic acid probes eliminate the need for multi-layer detection procedures (e.g., antibody-based-systems), which allows fast processing and also reduces non-specific background signals. Therefore, fluorescence in situ hybridization (FISH) has become an increasingly popular and valuable tool in both basic and clinical sciences.
Because of the importance of FISH technology in molecular biology and cytogenetics, optimizing current FISH technology to improve the sensitivity of hybridization (fluorescence) signals, to simplify and thus decrease the time for the process, and to substitute toxic reagents with non-health-hazard chemicals used in the FISH process is desirable. FISH technology for DNA (or RNA) chromosomes is dependent on four major factors: (a) optimal temperature for effective denaturation of double-strand DNAs (separation of two DNA strands); (b) optimal temperature for annealing or hybridization between target DNA (or RNA) and labeled DNA or RNA probes (i.e., DNA or anti-sense RNA fragments with which enzymes, fluorochromes, chromophores, chemiluminescers, bioluminescers, radioisotopes, biotin or avidin are conjugated); (c) selection of suitable solutions to enhance both the denaturation and the hybridization processes; and (d) effective post-hybridization washing conditions. It is essential that the structural integrity of nuclei, chromosomes, cells, tissue sections and spatial resolution of the fluorescence signals not be compromised during the FISH process. Therefore, optimization of FISH technology should include increased hybridization efficiency, increased detection sensitivity, and preservation of cellular, tissue, nuclear, and chromosomal morphology.
Currently, FISH procedures performed by many laboratories around the world are generally very similar to those of Kuo, et al., ("Detection of Aneuploidy Involving Chromosomes 13, 18 or 21, by Fluorescence in Situ Hybridization to Interphase and Metaphase Amniocytes," Am. J. Hum. Genet. 49:112-119 (1991)); Klinger, et al., ("Rapid Detection of Chromosome Aneuploidies in Uncultured Amniocytes by Using Fluorescence in Situ Hybridization (FISH)," Am J. Hum. Genet. 51:55-65 (1992)); and Ward, B. E., et al., ("Rapid Prenatal Diagnosis of Chromosomal Aneuploidies by Fluorescence in Situ Hybridization; Clinical Experience with 4,500 Specimens," Am. J. Hum. Genet. 52:854-865 (1993)). However, most laboratories rely, for convenience on kits available from two major commercial sources: Oncor, Inc., "Rapid Chromosome In Situ Hybridization System", Edition 1, October 1993; and Vysis, Inc. Biological Detection Systems/Amersham, Inc., provides only labeled DNA probes with a recommended FISH protocol (Biological Detection Systems, Inc., 955 William Pitt Way, Pittsburgh, Pa. 15238). All of these FISH procedures are time consuming, labor intensive, extremely tedious, and of very limited detection sensitivity. U.S. Pat. No. 5,225,326, to Bresser, et al., teaches "one step in situ hybridization," wherein both fixation and FISH can purportedly be performed in 5 minutes to 4 hours. Haar, et al., "A Rapid FISH Technique for Quantitative Microscopy," Bio Techniques, Vol. 17, No. 2, pp. 346-353 (August 1994), discloses a technique which can "in principle" reduce the time necessary for FISH to thirty minutes.
Scoring fluorescence signals using the FISH procedures described above generally requires a 100.times. oil-immersion objective lens with a triple bandpass filter due to lower signal sensitivity. The use of a high concentration of formamide during the FISH process appears to incur morphological destruction of cellular, nuclear or chromosomal structure. Furthermore, all of these processes involve the use of formamide during hybridization or the post-hybridization process. Formamide is an expensive, toxic solvent and also a teratogen. Therefore, a formamide-free FISH process is environmentally and hygienically desirable.